Osteoarthritis of the equine distal interphalangeal joint is a common cause of lameness. MicroRNAs from biofluids are promising biomarkers and therapeutic candidates. Synovial fluid samples from horses with mild and severe equine distal interphalangeal joint osteoarthritis were submitted for small RNA sequencing. The results demonstrated that miR-92a was downregulated in equine synovial fluid from horses with severe osteoarthritis and there was a significant increase in COMP, COL1A2, RUNX2 and SOX9 following miR-92a mimic treatment of equine chondrocytes in monolayer culture. This is the first equine study to evaluate the role of miR-92a in osteoarthritic chondrocytes in vitro.

Tooth resorption (TR) in domestic cats is a common and painful disease characterised by the loss of mineralised tissues from the tooth. Due to its progressive nature and unclear aetiology the only treatment currently available is to extract affected teeth. To gain insight into TR pathogenesis, we characterised the transcriptomic changes involved in feline TR by sequencing RNA extracted from 14 teeth (7 with and 7 without signs of resorption) collected from 11 cats. A paired comparison of teeth from the same cat with and without signs of resorption identified 1,732 differentially expressed genes, many of which were characteristic of osteoclast activity and differentiation, in particular matrix metalloproteinase 9 (MMP9). MMP9 expression was confirmed by qPCR and immunocytochemistry of odontoclasts located in TR lesions. A hydroxamate-based MMP9 inhibitor reduced both osteoclast formation and resorption activity while siRNA targeting MMP9 also inhibited osteoclast differentiation although had little effect on resorption activity. Overall, these results suggest that increased MMP9 expression is involved in the progress of TR pathogenesis and that MMP9 may be a potential therapeutic target in feline TR.

Despite many recent advances in imaging and epidemiological data analysis, musculoskeletal injuries continue to be a welfare issue in racehorses. Peptide biomarker studies have failed to consistently predict bone injury. Molecular profiling studies provide an opportunity to study equine musculoskeletal disease. A systematic review of the literature was performed using preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P) guidelines to assess the use of miRNA profiling studies in equine and human musculoskeletal injuries. Data were extracted from 40 papers between 2008 and 2020. Three miRNA studies profiling equine musculoskeletal disease were identified, none of which related to equine stress fractures. Eleven papers studied miRNA profiles in osteoporotic human patients with fractures, but differentially expressed miRNAs were not consistent between studies. MicroRNA target prediction programmes also produced conflicting results between studies. Exercise affected miRNA profiles in both horse and human studies (e.g., miR-21 was upregulated by endurance exercise and miR-125b was downregulated by exercise). MicroRNA profiling studies in horses continue to emerge, but as yet, no miRNA profile can reliably predict the occurrence of fractures. It is very important that future studies are well designed to mitigate the effects of variation in sample size, exercise and normalisation methods.

Advanced next generation sequencing approaches have started to reveal the cellular and molecular complexity of the microenvironment in many tissues. It is challenging to obtain high quality RNA from mineralised tissues. We developed an optimised method of RNA extraction from feline teeth collected in a clinical setting and at post mortem. Teeth were homogenised in phenol-guanidinium solution at near-freezing temperatures and followed by solid-phase nucleic acid extraction utilising a commercially available kit. This method produced good RNA yields and improved RNA quality based on RNA integrity numbers equivalent (RIN^e) from an average of 3.6 to 5.6. No correlation was found between RNA purity parameters measured by A_260:280 or A_230:260 ratios and degree of RNA degradation. This implies that RNA purity indicators cannot be reliably used as parameters of RNA integrity. Two reference genes (GAPDH, RPS19) showed significant changes in expression levels by qPCR at low and moderate RIN^e values, while RPL17 was stable at all RIN^e values tested. Furthermore, we investigated the effect of quantity and quality of RNA on the quality of the resultant RNA sequencing (RNA-Seq) data. Thirteen RNA-seq data showed similar duplication and mapping rates (94 to 95%) against the feline genome regardless of RIN^e values. However one low yield sample with a high RIN^e value showed a high duplication rate and it was an outlier on the RNA-seq multidimensional scaling plot. We conclude that the overall yield of RNA was more important than quality of RNA for RNA-seq quality control. These results will guide researchers who wish to perform RNA extractions from mineralised tissues, especially if collecting in a clinical setting with the recognised restraints that this imposes.

The authors regret that the original version of the above article contained errors in the Figs. 3, 4 and Tables 3 legends. The errors has been corrected.